The primary objective of this proposal is to understand how proteins are imported into mitochondria. Most mitochondrial proteins are encoded in the nucleus, synthesized in the cytoplasm, and post-translationally imported into the organelle. Newly synthesized mitochondrial proteins must find the mitochondrion, cross one or both of the mitochondrial membranes, and be sorted to the correct submitochondrial compartment. The primary goal of this proposal is to identify new membrane components of the import pathway. Although a number o proteins required for mitochondrial protein import have been recently identified, many critical proteins, such as components of the protein-translocating pore or channel, have yet to be discovered. Furthermore, most of the machinery required to sort imported proteins to specific submitochondrial compartments is likewise unidentified. Using two approaches with the yeast, Saccharomyces cerevisiae, we have identified new import components, and we are analyzing their role in the mitochondrial protein import and sorting. Determine the Function of MAS6, an Import Component of the Mitochondrial Inner Membrane mas6, a previously uncharacterized import mutant, is defective at an early step in the import pathway. Recently, we have shown that MAS6 encodes an essential 23kD protein localized in the mitochondrial inner membrane, and enriched in contact sites. Hence MAS6 represents the first import component identified in the inner membrane. To determine the precise role the MAS6 protein plays in the import pathway, we propose to extend our investigations of MAS6. For example, we will analyze the import or precursors into mitochondria isolated from different mas6 mutants. We will also ask if MAS6 directly interacts with an imported precursor, and determine if MAS6 is member of a complex or import proteins located in the inner membrane. We also propose to use MAS6 to identify new import components of the inner membrane. Identification and Analysis of Membrane Proteins Required to Sort Proteins to the Mitochondrial Intermembrane Space. Many proteins destined for the mitochondrial intermembrane space (IMS) are proposed to be imported first completely into the matrix, and then exported back across the inner membrane by a process analogous to bacterial secretion. To identify the machinery required for IMS sorting, we have selected mutants that mislocalize an IMS-bound fusion protein. These mutants have been used to precisely map the signal required for export across the inner membrane. We have also isolated a number of mutants that are defective in components of the export pathway. We propose to use these mutants to identify membrane proteins that mediate IMS sorting, and determine their function in import.